Songklanakarin J. Sci. Technol. 35 (3), 317-323, May - Jun. 2013 http://www.sjst.psu.ac.th Original Article Inhibition of nitric oxide production by compounds from Boesenbergia longiflora using lipopolysaccharide-stimulated RAW264.7 macrophage cells Teeratad Sudsai1,2, Chatchai Wattanapiromsakul1, and Supinya Tewtrakul1, 2* 1Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, 2Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 90112 Thailand. Received 12 October 2012; Accepted 17 January 2013 Abstract The inhibitory activity of extract and compounds isolated from Boesenbergia longiflora against nitric oxide (NO) was evaluated using RAW264.7 cells. Isolation of the chloroform extract of B. longiflora rhizomes afforded four known flavonoids, which were identified as kaempferol-3,7,4'-trimethyl ether (1), kaempferol-7,4'-dimethyl ether (2), rhamnazin (3), pinostrobin (4), together with four known diarylheptanoids, dihydrobisdemethoxycurcumin (5), curcumin (6), demethoxy- curcumin (7) and bisdemethoxycurcumin (8), as well as one sterol, -sitosterol-D-glucoside (9). Compound 6 exhibited the highest inhibitory activity against NO release with an IC50 value of 4.5 µM, followed by 7 (IC50 = 11.7 µM), 8 (IC50 = 15.7 µM), 5 (IC50 = 23.0 µM) and 1 (IC50 = 23.5 µM), respectively. This study demonstrated that diarylheptanoids and some methoxy- flavonoids found in B. longiflora are responsible for anti-inflammatory activity and this is the first report the safety, chemical constituents and biological activity of this plant. Keywords: Zingiberaceae, anti-inflammatory effect, diarylheptanoids, methoxyflavonoids 1. Introduction matory, anticancer, and anti-HIV activities. Cyclohexenyl chalcone derivatives, 4-hydroxypanduratin A and panduratin The genus Boesenbergia, a member of the A, the active constituents isolated from B. rotunda, exhibited Zingiberaceae family, is distributed from India to Southeast inhibitory activities on dengue 2 virus NS3 protease (Kiat et Asia with approximately 80 species worldwide, where 19 al., 2006) and HIV-1 protease (Cheenpracha et al., 2006) as species were previously accounted for Thailand (Delin and well as anti-inflamatory activity (Tuchinda et al., 2002; Larsen, 2000; Techaprasan et al., 2006). Many researchers Tewtrakul et al., 2009). Due to these properties, Boesenbergia have demonstrated several pharmacological properties of species have gained attention as important sources for plants in the Zingiberaceae family, however, studies that medicinal treatment. describe the effects of Boesenbergia species are still limited. Nitric oxide (NO) is one of pro-inflammatory mediators Only Boesenbergia rotunda has been extensively reported released in response to pathogenic infections. During the as having several pharmacological and phytochemical prop- inflammatory process, macrophages generate NO to eliminate erties ranging from antimicrobial, antioxidant, anti-inflam- foreign pathogens, recruit other cells to the infected area and subsequently resolve the inflammation. In general, NO is involved in vasodilation, platelet aggregation, host defense * Corresponding author. and a regulatory molecule with homeostatic activities Email address: [email protected], [email protected] (Korhonen et al., 2005; Min et al., 2009). However, excessive 318 T. Sudsai et al. / Songklanakarin J. Sci. Technol. 35 (3), 317-323, 2013 production of NO also attacks normal tissue surrounding the SKP2060200-301 and SKP206111601 respectively. The plant infected area by binding with other superoxide radicals and materials were identified by Dr. Charun Marknoi, Queen Sirikit acts as a reactive radical which damages normal cell function. Botanical Garden, Chiang Mai, Thailand. The voucher speci- NO is synthesized from L-arginine via the catalytic action of mens are kept at the herbarium of the Faculty of Pharma- the enzyme, the nitric oxide synthase (NOS). Inducible nitric ceutical Sciences, Prince of Songkla University, Songkhla, oxide synthase (iNOS) is responsible for the over- production Thailand. The plant material was cleaned with distilled water. of NO which is known to be involved in various chronic Only fresh rhizomes, which did not have scars or spots of inflammatory diseases, including cancer. Lipopolysaccharide disease, were selected for further extraction. The rhizomes (LPS) from Gram negative bacteria is particularly well known were cut into round slices in appropriate sizes of 3-5 mm to increase iNOS expression and over-production of NO, transverse sections. The rhizomes were then dried in a hot leading to the initiation of an inflammatory response (Tew- air oven at 50°C until dryness. The dried rhizomes were trakul et al., 2009; Min et al., 2009). Thus, the inhibition of pulverized to coarse powder using an electric blender. NO production is an important therapeutic consideration in the development of anti-inflammatory agents and treatment 2.3 Preparation of the plant extracts of inflammatory disorders. Boesenbergia longiflora (Wall.) Kuntze is locally Ten grams of each dried plant was extracted by reflux known in Thai as Wan priao. Its rhizome has been used in for 3 h with 200 ml of ethanol (EtOH) and water, separately. ethnomedical uses as tonic (Chuakul and Boonpleng, 2003). The solvents were removed under reduced pressure to give Neither phytochemical nor pharmacological studies have ethanolic and water extracts, respectively. The yields of been previously reported for this plant species. In addition, ethanolic extracts of K. rotunda, K. angustifolia, K. margi- the ethanol and water extracts from the rhizomes of B. nata, K. pulchra, Kaempferia sp., B. thorelii, B. longiflora, longiflora exhibited anti-inflammatory activity in murine Boesenbergia sp. 1 and Boesenbergia sp. 2 were found to be macrophage-like RAW264.7 cell line. We therefore, investi- 18.5, 11.6, 31.7, 10.1, 21.6, 15.4, 14.9, 9.8 and 13.1 %w/w, gated the inhibitory activity of compounds isolated from B. respectively, whereas the water extracts were 44.9, 25.3, 14.1, longiflora on LPS-induced NO release using RAW264.7 cells. 10.2, 16.3, 11.6, 20.5, 12.5 and 37.2 %w/w, respectively. Stock solutions (10 mg/ml) of the extracts were prepared in DMSO 2. Materials and Methods and stored at 4°C until use. 2.1 Reagents 2.4 Extraction and isolation of compounds from B. longiflora Lipopolysaccharide (LPS) from Escherichia coli, The fresh rhizomes of B. longiflora (20 kg, second RPMI-1640 medium, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- lot) were sliced and oven-dried at 50°C. Five kilograms of 2H-tetrazolium bromide (MTT), ibuprofen, aspirin, L-nitro- pulverized rhizomes were macerated with EtOH at room arginine (L-NA), caffeic acid phenethyl ester (CAPE), temperature, four times (6 L × 4). The EtOH extract (416 g) was indomethacin, phosphate buffer saline (PBS) and tween 80 then concentrated and partitioned between 95% MeOH and were from Sigma Aldrich (Sigma Aldrich, Missouri, USA). hexane, and successively partitioned with chloroform and Fetal calf serum (FCS) was from Gibco (Invitrogen, California, water. After that, the water layer was partitioned with ethyl USA). Penicillin-streptomycin was from Invitrogen acetate (EtOAc). Each partition was evaporated to dryness in (Invitrogen, California, USA). 96-Well microplates were vacuo to give residues of hexane (116.6 g), chloroform (285.6 obtained from Nunc (Nunc, Birkrød, Denmark). Other chemi- g), EtOAc (2.3 g) and water fractions (23.3 g), respectively. cals were from Sigma Aldrich (Sigma–Aldrich, Missouri, The chloroform fraction (30.0 g), which possessed the highest USA). NO inhibitory activity (IC50 = 5.5 µg/ml), was chromato- graphed on silica gel (800 g) using a stepwise gradient elution 2.2 Plant materials of hexane, hexane-chloroform (9:1, 8:2, 6:4, 4:6, 2:8), chloro- form, chloroform-EtOAc (9:1, 8:2, 6:4, 4:6, 2:8), EtOAc and The rhizomes of nine selected Zingiberaceous plants EtOAc-MeOH (9.5:0.5, 9:1), to give thirteen fractions (F1- including Kaempferia rotunda L., Kaempferia angustifolia F13). Roscoe, Kaempferia marginata Carey ex Roscoe, Kaempferia Fraction F6 (11.6 g) was chromatographed on silica sp. (Prauh-Pa), Boesenbergia thorelii (Gagnep.) Loes., gel (400 g) using a gradient of hexane, hexane-chloroform Boesenbergia longiflora, Boesenbergia sp. 1 (Kai-Dang) and (9:1, 8:2, 6:4, 4:6, 2:8), chloroform and chloroform-MeOH (9.5: Boesenbergia sp. 2 (Kai-Dam) were bought from Chatuchak 0.5, 9:1) solvent system, to give compound 1 (kaempferol-3,7, weekend market in Bangkok, Thailand, whereas Kaempferia 4'-trimethyl ether, 24 mg), together with compound 2 pulchra Ridl. rhizomes were collected in October 2010 in (kaempferol-7,4'-dimethyl ether, 32 mg). Subfraction of F6 Suratthani province, Thailand. The voucher specimens are (12.7 mg) was chromatograped on preparative thin layer SKP 206111801, SKP 206110101, SKP206111301, SKP 2061100- chromatographed using hexane-chloroform (1:1) as a de- 101, SKP206022001, SKP2060200-101, SKP2060200-201, veloping solvent, to yield compound 4 (pinostrobin, 3 mg). T. Sudsai et al. / Songklanakarin J. Sci. Technol. 35 (3), 317-323, 2013 319 Fraction F8 (235 mg) was chromatographed, using the same µg/ml) and 10% FCS in 75-cm2 culture flasks at 37°C in a solvent system as that of fraction 6, to give compound 3 humidified atmosphere containing 5% CO2. Cells were (rhamnazin, 2 mg) and compound 5 (dihydrobisdemethoxy- harvested with trypsin-EDTA